Biological membranes form the interface between cells and their environment, and transport of molecules across this barrier is essential for all cells. Whereas a wealth of knowledge exists about the transport of hydrophilic compounds, very little is known about membrane transport of hydrophobic molecules. This is remarkable, since biological membranes pose unique problems for the passage of hydrophobic molecules. The outer membrane (OM) of gram-negative bacteria is a particularly effective barrier against permeation of hydrophobic compounds, due to the presence of lipopolysaccharide (LPS). The only OM proteins that have been shown to transport hydrophobic compounds belong to the FadL transporter family. Members of this family are widespread in gram-negative bacteria and play a role in the uptake of long-chain fatty acids (LCFAs) and xenobiotics. The goal of this project is to understand in structural and mechanistic terms how such molecules are transported across the OM. More specifically, we will address the following questions: 1. Characterization of FadL-mediated LCFA transport. Using the crystal structures of E. coli FadL as a starting point, we will test and further delineate the proposed model of LCFA transport, by using a combination of site-directed mutagenesis, in vivo transport assays and X-ray crystallography. 2. Characterization of substrate specificity of FadL orthologues involved in LCFA and xenobiotics transport by using in vivo and in vitro substrate binding and transport assays. 3. Mechanism of xenobiotics transport by FadL orthologues. By solving crystal structures of xenobiotics transporters we will determine the structural basis for their substrate specificity. These structures will also allow us to determine whether mechanistic differences exist between LCFA and xenobiotics transport.